Part:BBa_K678002

eGFP-PTS1 for mammalian cells

This is a device intended for expression of green fluorescent protein (GFP) in the peroxisomes of mammalian cells. The expression of GFP is under the control of the strong constitutive cytomegalovirus (CMV) promoter. The targeting sequence S-K-L often referred to as peroxisomal targeting sequence 1 (PTS1) is directly fused to the C-terminal of GFP, this sequence should ensure the localization of GFP to the peroxisomes of the cell.

Usage and Biology

We used this device to target enhanced GFP to the peroxisomes of the mammalian cell line U-2 OS (Figure 1). Although the microscopy shows GFP distributed in points which most likely are the peroxisomes of the cells, further analysis are necessary to confirm that GFP is in fact is targeted to the peroxisomes. We furthermore transiently transfected U-2 OS with both the device contained on plasmid pJEJAM2 and with pJEJAM5 expressing cyan fluorescent protein (CFP) (Figure 2).

DTU-Denmark-2 2011 Figure 1: U-2 OS cells transiently transfected with pJEJAM2 expressing GFP localized to the peroxisomes. As can be seen on the picture this vector most likely localizes GFP to the peroxisomes of the cells. The white bar has a length of 30μm.

DTU-Denmark-2 2011 Figure 2: U-2 OS cells transiently transfected with pJEJAM2 and pJEJAM5 expressing GFP localized to the peroxisomes and CFP, respectively. The white bar has a length of 30μm.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2233
  Illegal SpeI site found at 49
  Illegal PstI site found at 2323
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2233
  Illegal SpeI site found at 49
  Illegal PstI site found at 2323
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2233
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2233
  Illegal SpeI site found at 49
  Illegal PstI site found at 2323
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2233
  Illegal SpeI site found at 49
  Illegal PstI site found at 2323
  Illegal NgoMIV site found at 3088
  
• 1000
  COMPATIBLE WITH RFC[1000]